The present method relates to the translational activation of genes using the ribosome recruitment protein, eIF4G or an eIF4G-like protein. The invention relates to the translation of RNA molecules containing heterologous protein-binding sites, which RNA molecules encode one, two, three or more proteins. The invention provides products and methods for the identification of RNA-binding proteins. The invention further provides a system by which proteinxe2x80x94protein interactions and inhibitors or enhancers of these interactions may be identified. Further, the invention provides products and methods to provide a cell with one or more therapeutic proteins. The invention provides products and methods for controlling the levels of translation of such proteins. The invention provides products and methods to control the translation and stoichiometry of multiple subunit proteins. The invention provides products for and methods of screening for proteins which interact with an RNA binding site, and methods for identifying RNA binding sites.
Citation of a reference herein shall not be construed as an admission that such is prior art to the present invention.
Proteins and proteinxe2x80x94protein interactions play a central role in the various essential biochemical processes. For example, these interactions are evident in the interaction of hormones with their respective receptors, in the intracellular and extracellular signaling events mediated by proteins, in enzyme substrate interactions, in intracellular protein trafficking, in the formation of complex structures like ribosomes, viral coat proteins, and filaments, and in antigen-antibody interactions. These interactions are usually facilitated by the interaction of small regions within the proteins that can fold independently of the rest of the protein. These independent units are called protein domains. Abnormal or disease states can be the direct result of aberrant proteinxe2x80x94protein interactions. For example, oncoproteins can cause cancer by interacting with and activating proteins responsible for cell division. Protein-protein interactions are also central to the mechanism of a virus recognizing its receptor on the cell surface as a prelude to infection proteinxe2x80x94protein interactions direct signal transduction cascades that result in a biological response. Identification of domains that interact with each other not only leads to a broader understanding of proteinxe2x80x94protein interactions, but also aids in the design of inhibitors of these interactions.
Protein-protein interactions have been studied by both biochemical and genetic methods. The biochemical methods are laborious and slow, often involving painstaking isolation, purification, sequencing and further biochemical characterization of the proteins being tested for interaction. As an alternative to the biochemical approaches, genetic approaches to detect proteinxe2x80x94protein interactions have gained in popularity as these methods allow the rapid detection of the domains involved in proteinxe2x80x94protein interactions.
An example of a genetic system to detect proteinxe2x80x94protein interactions is the xe2x80x9cTwo-Hybridxe2x80x9d system to detect proteinxe2x80x94protein interactions in the yeast Saccharomyces cerevisiae (Fields and Song, 1989, Nature 340:245-246; U.S. Pat. No. 5,283,173 by Fields and Song). This assay utilizes the reconstitution of a transcriptional activator like GAL4 (Johnston, 1987, Microbiol. Rev. 51:458-476) through the interaction of two protein domains that have been fused to the two functional units of the transcriptional activator: the DNA-binding domain and the activation domain. This is possible due to the bipartite nature of certain transcription factors like GAL4. Being characterized as bipartite signifies that the DNA-binding and activation functions reside in separate domains and can function in trans (Keegan et al., 1986, Science 231:699-704). The reconstitution of the transcriptional activator is monitored by the activation of a reporter gene such as the lacZ gene that is under the influence of a promoter that contains a binding site (Upstream Activating Sequence or UAS) for the DNA-binding domain of the transcriptional activator. This method is most commonly used either to detect an interaction between two known proteins (Fields and Song, 1989, Nature 340:245-246) or to identify interacting proteins from a population that would bind to a known protein (Durfee et al., 1993, Genes Dev. 7:555-569; Gyuris et al., 1993, Cell 75:791-803; Harper et al., 1993, Cell 75:805-816; Vojtek et al., 1993, Cell 74:205-214).
Another system that is similar to the Two-Hybrid system is the xe2x80x9cInteraction-Trap systemxe2x80x9d devised by Brent and colleagues (Gyuris et al., 1993, Cell 75:791-803). This system is similar to the Two-Hybrid system except that it uses a LEU2 reporter gene and a lacZ reporter gene. Thus proteinxe2x80x94protein interactions also lead to the reconstitution of the transcriptional activator system and allows cells to grow in media lacking leucine and enable them to express xcex2-galactosidase. The DNA-binding domain used in this system is the LexA DNA-binding domain, while the activator sequence is obtained from the B42 transcriptional activation domain (Ma and Ptashne, 1987, Cell 51:113-119). The promoters of the reporter genes contain LexA binding sequences and hence will be activated by the reconstitution of the transcriptional activator. Another feature of this system is that the gene encoding the DNA-binding domain fusion protein is under the influence of an inducible GAL promoter so that confirmatory tests can be performed under inducing and non-inducing conditions.
Still other versions of the two-hybrid approach exist, for example, a xe2x80x9cContingent Replication Assayxe2x80x9d has been reported (Nallur et al., 1993, Nucleic Acids Res. 21:3867-3873; Vasavada et al., 1991, Proc. Natl. Acad. Sci. USA 88:10686-10690). In this case, the reconstitution of the transcription factor in mammalian cells due to the interaction of the two fusion proteins leads to the activation of transcription of the SV40 T antigen. This antigen allows the replication of the activation domain fusion plasmids. Another modification of the two-hybrid approach using mammalian cells is the xe2x80x9cKaryoplasmic Interaction Selection Strategyxe2x80x9d that also uses the reconstitution of a transcriptional activator (Fearon et al., 1992, Proc. Natl. Acad. Sci. USA 89:7958-7962). Reporter genes used in this case have included the gene encoding the bacterial chloramphenicol acetyl transferase, the gene for cell-surface antigen CD4, and the gene encoding resistance to Hygromycin B. In both of the mammalian systems, the transcription factor that is reconstituted is a hybrid transcriptional activator in which the DNA-binding domain is from GAL4 and the activation domain is from VP16.
Recently, a transcriptional activation system has been described to isolate and catalog possible proteinxe2x80x94protein interactions within a population, and allow the comparison of such interactions between two populations (see PCT Publication WO 97/47763 published Dec. 18, 1997).
However, all of the assays mentioned above utilize a transcriptional activation system which examines the interaction of DNA binding proteins with DNA of a reporter gene. Additionally, the transcriptional systems require that proteins being assayed be driven into the nucleus. Accordingly, there is a need in the art for a system which allows for detecting proteinxe2x80x94protein interactions and inhibitors or enhancers of such interactions in the cytoplasmic compartment of a cell. The present invention provides such a system.
Additionally, none of the systems described above provide a means by which a protein important for the translational-activation of a gene may be identified. Nor do any of the methods described above provide a method for activating the translation of a gene-of-interest. The present invention provides such methods and compositions as well as therapeutic, diagnostic, and analytical uses of such methods and compositions.
Procaryotic and eucaryotic cells use different strategies to specify the translation start site on an mRNA molecule. In bacterial mRNAs a conserved stretch of six nucleotides, called the Shine-Dalgarno sequence, is located a few nucleotides upstream from the initiating AUG codon. This sequence pairs with the 16S RNA in the small ribosomal subunit and thereby correctly positions the initiating AUG codon in the ribosome. This interaction controls the efficiency of initiation of translation in bacteria, and many of the translational control systems in procaryotes involve blocking the Shine-Dalgarno sequence by covering the sequence with a protein or by incorporating it into a base-paired region in the mRNA molecule.
In contrast, eucaryotic mRNAs do not contain a Shine-Dalgarno sequence. In eucaryotes, the selection of an AUG as a translational start site has been thought to be determined by the proximity of the AUG to the cap at the 5xe2x80x2 end of the mRNA molecule, where the small ribosomal subunit binds to the mRNA and begins scanning for an initiating AUG codon (In Molecular Biology of the Cell, 3d ed., 1994, Alberts, B. et al. pp. 461-468). If the recognition of the AUG codon is poor, the scanning ribosomal subunits will ignore the first AUG in the mRNA and skip to the second or third AUG codon, Id at 461. The result of this xe2x80x9cleaky scanningxe2x80x9d process is to produce two or more proteins from the same mRNA that differ in their amino termini. However, a majority of eukaryotic genes begin translation at the first AUG codon, Id. at 461.
Another significant difference between procaryotic and eucaryotic translation is that the eucaryotic ribosomes dissociate rapidly from mRNA when translation terminates, Id. at 462. Accordingly, reinitiation at an internal AUG codon is less efficient in eucaryotes than procaryotes. This difference serves to explain why a majority of eucaryotic mRNAs encode only a single protein that is translated from the first AUG from the 5xe2x80x2 end of the mRNA molecule, Id. at 462.
Some eucaryotic cell and viral mRNAs can initiate translation by an alternative mechanism that involves internal initiation rather than scanning. These mRNAs contain complex nucleic acid sequences called internal ribosome entry sites (IRES) Id. at 462. IRES bind ribosomes in a cap independent manner (see. Section 2.3), and translations begins at an AUG codon that is 3xe2x80x2 to the entry site.
Translation of most eukaryotic mRNAs requires a 5xe2x80x2 cap structure (m7GpppN) and 3xe2x80x2 poly(A) tail (Gallie, D., 1991, Genes and Dev. 5:2108-2116). These structures promote translation initiation by binding to the eukaryotic translation initiation factor (eIF4E) and the poly(A)-binding protein (PABP), respectively. The protein eIF4G forms a molecular bridge between eIF4E (Mader, et al., 1995, Mol. Cell. Biol. 15:4990-4997) and PABP (Tarun, et al., 1996, EMBO J. 15:7168-7177, Imataka, et al., 1998, EMBO J. 17:7480-7489). Binding of eIF4G leads to a circularizing of the mRNA (Wells, et al., 1998, Mol. Cell 2:135-40). eIF4G also binds the 40S ribosomal subunit via eIF3 (Lamphear, et al., 1995, J. Biol. Chem. 270:21975-21983).
The binding of the small ribosomal subunit as part of the 43S translation pre-initiation complex represents a rate-limiting step in mRNA translation (Sachs, et al., 1997, Cell 89:831-838). The 5xe2x80x2 cap structure and the 3xe2x80x2 poly(A) tail with their respective binding proteins have been shown to play critical roles (Gallie, D., 1991, Genes and Dev. 5:2108-2116, Tarun, et al., 1996, EMBO J. 15:7168-7177; Preiss, T., et al., 1998, Nature 392:516-520). The function of eIF4G in ribosome recruitment is less clearly defined (Hentze, et al., 1997, Science 275:500-501). eIF4G is a subunit of the cap binding complex, which complex also includes eIF4F and the cap recognition factor eIF4E and, in higher eukaryotes, the RNA-dependent ATPase eIF4A (FIG. 1a, upper scheme). Stimulated by eIF4B, eIF4A is thought to unwind secondary structure in the 5xe2x80x2 UTR of the mRNA. eIF4G has a modular structure (see FIG. 3a). It interacts with eIF4E (Mader, et al., 1995, Mol. Cell. Biol. 15:4990-4997) and PABP (Imataka, et al., 1998, EMBO J. 17:7480-7489). The central region bears a putative RNA recognition motif (RRM) (Goyer, C. et al., 1993, Mol. Cell. Biol. 13:4860-4874, De Gregorio, et al., 1998, RNA 4:828-36) and binding sites for eIF4A (Imataka, et al., 1997, Mol. Cell. Biol. 17:6940-6947) and eIF3 (Lamphear, et al. 1995, J. Biol. Chem. 270:21975-21983). The C-terminal harbors a second binding site for eIF4A (Lamphear, et al., 1995, J. Biol. Chem. 270:21975-21983) and for the eIF4E kinase Mnk1 (Pyronnet, S. et al., 1999, EMBO J. 13:270-279).
As described herein, the inventors of the present invention have made the surprising discovery that a core region of human eIF4G1 (in the example, amino acids 642-1091), lacking both the eIF4E- and PABP-binding sites functions as an autonomous ribosome recruitment core in vivo. Further the inventors demonstrate that fusion of this region of eIF4G1 to the IRE-binding protein IRP-1 suffices to direct the translation of downstream cistron of bicistronic or multi-cistronic mRNAs bearing IREs in their intercistronic space. This function of translational activation is preserved even when translation via the 5xe2x80x2 end is inhibited. Thus, eIF4G-like proteins (including but not limited to mammalian eIF4G1) have been discovered to represent the critical ribosome recruitment factor sufficient to drive downstream translation in vivo.
Accordingly, the present invention provides methods and means to detect and isolate the genes encoding RNA-binding proteins. The invention provides methods for detecting binding sites in an RNA molecule for such proteins. The methods of the invention provide the reconstitution of a selectable event, which is the formation of a translation factor. In one embodiment, the reconstitution of a translation factor occurs by interaction of fusion proteins expressed by chimeric genes. In a preferred embodiment, the type of fusion protein used is an RNA-binding protein fused to eIF4G-like protein or a translationally active derivative of an eIF4G-like protein. In another embodiment, RNA-binding proteins are found by fusing eIF4G-like protein or a translationally active derivative of eIF4G-like protein to a cDNA library. In a highly preferred embodiment, the fusion protein(s) drives translation of one, or more open reading frames from a downstream coding region of a bi-cistronic mRNA. In a most preferred embodiment, the fusion protein(s) of the invention drives the translation of several (e.g., two, three, four, five, six, seven, or more, etc.) open reading frames from a multi-cistronic mRNA. In a further embodiment, the level of translation (e.g., the amount of protein translated from each AUG start sites of a multi-cistronic RNA) is controlled by the use of protein-binding sites in the RNA (e.g., a heterologous protein-binding site (HBS)). In a further embodiment, the level of translation of each cistron is controlled by the use of different protein-binding sites in the RNA (e.g., different HBSs). In another embodiment, the level of translation is controlled by the number of HBSs that are placed intercistronically. In yet another embodiment, the level of translation is controlled by the proximity of an HBS to an adjacent and downstream cistron (e.g., the distance in nucleotides between the HBS and the cistron).
In yet another use of the invention, the reconstitution of a translational activator leads to the translation of a reporter protein which can be used to determine proteinxe2x80x94protein interactions. Not by way of limitation, the translational activator is reconstituted due to the proximity of the RNA-binding protein and the ribosome recruitment core (e.g., translationally active) of an eIF4G-like protein via the interaction of two test proteins. This reconstitution causes translation of reporter genes or downstream cistron(s) that, by way of example, contain a label or enable cells to grow in selective media. In a preferred aspect, the activity of a reporter gene is monitored enzymatically. The isolation of the plasmids that encode these fusion genes containing test proteins leads to the identification of the genes that encode proteins that interact with each other. In a specific embodiment, one of the test proteins of a proteinxe2x80x94protein interaction is known. In another embodiment, neither of the test proteins are known. In a further embodiment of the invention, inhibitors of a proteinxe2x80x94protein interaction are identified, by the lack of or decrease in translation of a reporter protein relative to that observed in the absence of the candidate inhibitor. In another embodiment, enhancers or facilitators of a proteinxe2x80x94protein interaction are identified, by the increase in translation of a reporter protein relative to that observed in the absence of the candidate facilitator.
Accordingly, this invention provides genetic and biochemical methods to identify and isolate proteins which interact. The invention also provides methods to identify proteins which bind to RNA. The present invention provides methods to identify RNA sequences to which an RNA-binding protein interacts.
The invention also provides methods to identify and isolate in a rapid manner the genes encoding the proteins involved in interactions that are specific to translational control of a gene. This invention provides methods for the identification of proteinxe2x80x94protein interactions that characterize a given population. This invention provides methods for the concurrent identification of inhibitors of the proteinxe2x80x94protein interactions. The invention further provides methods for controlling stoichiometry of multi-subunit proteins, and methods to produce one or more protein(s) in a host cell.
The invention relates to nucleic acids encoding a RNA comprising one or more heterologous protein-binding sites (e.g., HBS) and one or more genes. The invention also relates to recombinant cells containing a nucleotide sequence encoding a RNA containing a HBS. The invention further relates to nucleic acids encoding an eIF4G-like protein or derivatives or fragments thereof fused to a RNA-binding protein. The invention also relates to nucleic acids encoding an RNA-binding protein or derivatives fused to a first test protein, and an eIF4G-like protein fused to a second test protein and methods for reconstituting a translational activator.
The invention provides a nucleic acid encoding an RNA, said RNA comprising a coding region with one or more heterologous protein-binding sites in a non-coding region 5xe2x80x2 and adjacent to the coding region. In one embodiment the DNA molecule is purified. In another embodiment, the binding site is selected from the group consisting of IRE, MS2 RNA replicase site, U1A snRNA site, and xcex box B site.
The invention provides a DNA molecule comprising a promoter operably linked to a nucleotide sequence, which nucleotide sequence is transcribed in an appropriate cell to produce an RNA, said RNA comprising one or more coding regions, each with one or more heterologous protein-binding sites in a non-coding region 5xe2x80x2 and adjacent to the coding region. In one embodiment the RNA comprises two or more coding regions, and wherein a heterologous protein-binding site is in an intercistronic region. In one embodiment, at least one downstream coding region that is 3xe2x80x2 to another coding region is a reporter gene coding region. In another embodiment, at least one downstream coding region that is 3xe2x80x2 to another coding region encodes a Therapeutic. In yet another embodiment, at least two coding regions (a) are 3xe2x80x2 to another coding region, and (b) each encodes a different subunit of a multi-subunit protein. In another embodiment, the DNA molecule has two or more heterologous protein-binding sites in at least one intercistronic region. In yet another embodiment, the promoter is inducible.
In one embodiment, the invention provides expression vector comprising the DNA molecule comprising a promoter operably linked to a nucleotide sequence, which nucleotide sequence is transcribed in an appropriate cell to produce an RNA, said RNA comprising one or more coding regions, each with one or more heterologous protein-binding sites in a non-coding region 5xe2x80x2 and adjacent to the coding region; and an origin of replication. In one embodiment the expression vector is a plasmid.
The invention provides an RNA molecule comprising a coding region with a heterologous protein-binding site in a non-coding region 5xe2x80x2 and adjacent to the coding region. In one embodiment the RNA comprises two or more coding regions, and wherein a heterologous protein-binding site is in an intercistronic region. In another embodiment, the RNA molecule is purified.
The invention provides fusion protein comprising an RNA-binding protein fused to an eIF4G-like protein or a translationally active derivative thereof. In one embodiment the RNA-binding protein is fused to a translationally active derivative of a eIF4G-like protein. In a further embodiment, the translationally active derivative comprises an eIF3 binding domain of eIF4G1 . In another embodiment, the translationally active derivative lacks one or more of the PABP domain and the eIF4E binding domain. The invention provides nucleotide sequence encoding the fusion protein, and expression vectors comprising such sequence.
The invention provides a fusion protein comprising an eIF4G-like protein or translationally active derivative thereof fused to a second, different protein. In one embodiment the translationally active derivative of the eIF4G-like protein is fused to the second protein. In another embodiment the the translationally active derivative comprises an eIF3 binding domain of eIF4G1. In yet another embodiment, the translationally active derivative lacks one or more of the PABP domain and the eIF4E binding domain. The invention provides nucleotide sequence encoding the fusion protein, and expression vectors comprising such sequence.
The invention provides a fusion protein comprising an RNA-binding protein fused to a second, different protein. In one embodiment the RNA-binding protein is selected from the group consisting of IRP- 1, bacteriophage MS2 coat protein, spliceosomal protein U1A, and xcex box B binding protein. The invention provides nucleotide sequence encoding the fusion protein, and expression vectors comprising such sequence.
The invention provides a population of nucleic acids, wherein each nucleic acid in the population is a vector comprising (a) an origin of replication; (b) a nucleotide sequence encoding the fusion comprising an eIF4G-like protein or translationally active derivative thereof fused to a second, different protein; and (c) a promoter operably linked to said nucleotide sequence; wherein the identity of said second, different protein varies among said population. In one embodiment the population has a complexity of at least 100. In another embodiment the nucleotide sequences are those of a cDNA library. In another embodiment, the nucleotide sequences are of a random or biased peptide expression library.
The invention provides a population of nucleic acids, wherein each nucleic acid in the population is a vector comprising (a) an origin of replication; (b) a nucleotide sequence encoding the fusion comprising an RNA-binding protein fused to a second, different protein; and (c) a promoter operably linked to said nucleotide sequence; wherein the identity of said second, different protein varies among said population. In one embodiment the population has a complexity of at least 100. In another embodiment the nucleotide sequences are those of a cDNA library. In another embodiment, the nucleotide sequences are of a random or biased peptide expression library. The invention provides recombinant cells comprising the above nucleic acid. The invention provides transgenic organisms comprising as a transgene the above nucleic acids.
The invention provides a population of recombinant cells comprising the population of nucleic acids described above.
The invention provides methods of producing the above fusion proteins comprising subjecting a recombinant cell comprising the above nucleic acid to conditions such that the nucleotide sequence is expressed by the cell.
The invention provides kits comprising in one or more containers the nucleic acid the above-described nucleic acids.
The invention provides a nucleic acid comprising (a) a nucleotide sequence encoding an eIF4G-like protein or a translationally active derivative thereof; and (b) a polylinker region 5xe2x80x2 or 3xe2x80x2 to said nucleotide sequence that allows for insertion after restriction enzyme digestion of a nucleic acid fragment in the correct reading frame so as to encode a fusion protein to the eIF4G-like protein or derivative.
The invention provides a nucleic acid comprising (a) a nucleotide sequence encoding an RNA-binding protein; and (b) a polylinker region 5xe2x80x2 or 3xe2x80x2 to said nucleotide sequence that allows for insertion after restriction enzyme digestion of a nucleic acid fragment in the correct reading frame so as to encode a fusion protein to the RNA-binding protein.
The invention provides a method of producing a protein comprising contacting within a eukaryotic cell: (a) an RNA molecule comprising (i) a coding region encoding said protein, and (ii) a protein-binding site in a noncoding region 5xe2x80x2 and adjacent to said coding region; and (b) a fusion protein comprising (i) an RNA-binding protein that binds to said protein-binding site, fused to an eIF4G-like protein or a translationally active derivative thereof. In one embodiment the RNA molecule comprises two or more coding regions, and wherein a heterologous protein-binding site is in an intercistronic region, or has two or more heterologous protein-binding sites in at least one intercistronic region.
In another embodiment at least two coding regions (a) are 3xe2x80x2 to another coding region, and (b) each encodes a different subunit of a multi-subunit protein.
The invention provides a method of producing a protein comprising recombinantly expressing a fusion protein within a eukaryotic cell, wherein the cell contains a DNA molecule that is transcribed within the cell to produce a monocistronic or multicistronic RNA containing a heterologous protein-binding site in a region 5xe2x80x2 and adjacent to a coding region encoding said protein; wherein the fusion protein comprises (i) an RNA-binding protein that binds to said protein-binding site, fused to an eIF4G-like protein or a translationally active derivative thereof. In one embodiment the DNA molecule is a plasmid expression vector. In one embodiment the plasmid comprises an inducible promoter controlling production of said RNA. In another embodiment the fusion protein is expressed from a plasmid expression vector comprising a promoter operably linked to a nucleotide sequence encoding said fusion protein. In yet another embodiment the two or more identical heterologous protein-binding sites are in said intercistronic region. In still another embodiment, two or more intercistronic regions contain the heterologous protein-binding site, each of said two or more intercistronic regions encoding a different subunit of a multi-subunit protein.
The invention provides a method for detecting an RNA-binding protein comprising: (a) recombinantly expressing in a eukaryotic cell a fusion protein comprising an eIF4G-like protein or a translationally active derivative thereof fused to a test protein, wherein the cell comprises a DNA that is transcribed to produce a monocistronic or multicistronic RNA containing a heterologous protein-binding site in a region 5xe2x80x2 and adjacent to a reporter gene coding region; and (b) detecting an increase in the amount of the protein encoded by said reporter gene coding sequence, relative to said amount produced in the absence of said test protein, wherein an increase in said amount indicates that the test protein is an RNA-binding protein that binds to said heterologous protein-binding site. In one embodiment the two or more identical heterologous protein-binding sites are in said intercistronic region.
The invention provides a method for detecting a protein-binding site in an RNA comprising: (a) recombinantly producing in a eukaryotic cell: (i) a fusion protein comprising an eIF4G-like protein or a translationally active derivative thereof fused to a first protein for which it is desired to identify an RNA site to which said protein binds; (ii) a monocistronic or multicistronic RNA containing a heterologous test RNA sequence in a region 5xe2x80x2 and adjacent to a reporter gene coding region; and (b) detecting an increase in the amount of the protein encoded by said reporter gene coding sequence relative to said amount produced in the absence of said RNA sequence, wherein an increase in said amount indicates that the test RNA sequence is a protein-binding site that binds to said first protein. In one embodiment said fusion protein is expressed from an expression vector.
A method for detecting an RNA binding protein comprising: (a) recombinantly expressing within a population of eukaryotic cells a population of fusion proteins, each fusion protein comprising an eIF4G-like protein or a translationally active derivative thereof fused to a test protein, wherein the test protein varies among said population, wherein the cells comprise a DNA that is transcribed to produce a monocistronic or multicistronic RNA containing a heterologous protein-binding site in a region 5xe2x80x2 and adjacent to a reporter gene coding region; and (b) identifying a cell within said population that displays an increase in the amount of the protein encoded by said reporter gene relative to said amount produced in the absence of said test protein or in the presence of other fusion proteins, thereby identifying a cell in which the test protein is an RNA-binding protein that binds to said heterologous protein-binding site. In one embodiment the test protein portion of said fusion proteins are encoded by nucleotide sequences of a cDNA library. In another embodiment the fusion proteins are produced from plasmid expression vectors under the control of an inducible promoter.
The invention provides a method for detecting binding between a first test protein and a second test protein comprising: (a) recombinantly expressing in a eukaryotic cell (a) a first fusion protein comprising an eIF4G-like protein or a translationally active derivative thereof, fused to a first test protein; and (ii) a second fusion protein comprising an RNA-binding protein fused to a second test protein; wherein the cell comprises a DNA that is transcribed to produce a monocistronic or multicistronic RNA containing a heterologous protein-binding site in a region 5xe2x80x2 and adjacent to a reporting gene coding region, wherein said RNA-binding protein binds to said heterologous protein-binding site; and (b) detecting an increase in the amount of the protein encoded by said reporter gene coding region relative to said amount produced in the absence of one or both test proteins, wherein an increase in said amount indicates that the first test protein binds to said second test protein.
The invention provides a method for identifying a molecule that affects the amount of binding between a first protein and a second protein comprising: (a) recombinantly expressing in a eukaryotic cell in the presence of a candidate molecule (i) a first fusion protein comprising an eIF4G-like protein or a translationally active derivative thereof, fused to a first protein; and (ii) a second fusion protein comprising an RNA-binding protein fused to a second protein, wherein the first and second proteins bind to each other; wherein the cell comprises a DNA that is transcribed to produce a monocistronic or multicistronic RNA containing a heterologous protein-binding site in a region 5xe2x80x2 and adjacent to a reporter gene coding region, and wherein said RNA-binding protein binds to said heterologous protein-binding site; and (b) detecting an increase or decrease in the amount of the protein encoded by said reporter gene coding region relative to said amount produced in the absence of the candidate molecule, wherein said increase or decrease indicates that the candidate molecule inhibits or increases binding of said first protein to said second protein. In a specific embodiment, the candidate molecule is also recombinantly expressed in the cell.
The invention provides a method for identifying a molecule that complexes together a first protein and a second protein comprising: (a) recombinantly expressing in a eukaryotic cell in the presence of a candidate molecule (i) a first fusion protein comprising an eIF4G-like protein or a translationally active derivative thereof, fused to a first protein; and (ii) a second fusion protein comprising an RNA-binding protein, fused to a second protein, wherein the first and second proteins do not bind to each other; wherein the cell comprises a DNA that is transcribed to produce a monocistronic or multicistronic RNA containing a heterologous protein-binding site in a region 5xe2x80x2 and adjacent to a reporter gene coding region, wherein said RNA-binding protein binds to said heterologous protein-binding site; and (b) detecting an increase in the amount of the protein encoded by said reporter gene coding region relative to said amount produced in the absence of said candidate molecule, wherein said increase indicates that the candidate molecule complexes together said first protein and said second protein. In a specific embodiment, the candidate molecule is also recombinantly expressed in the cell. In one embodiment the candidate molecule is a candidate inhibitor molecule, and a decrease is detected in step (b), thereby indicating that the candidate molecule inhibits the binding of said first protein to said second protein. In another embodiment an increase is detected in step (b), thereby indicating that the candidate molecule increases the binding of said first protein to said second protein. In another embodiment step (a) comprises recombinantly expressing in a population of said cells a population of said first fusion proteins, wherein said first test protein varies among said population. In a further embodiment, said first test protein portions of said first fusion proteins are encoded by nucleotide sequences of a cDNA library. In another embodiment, step (a) comprises recombinantly expressing in a population of said cells a population of said second fusion proteins, wherein said second test protein varies among said population. In a further embodiment, said second test protein portions of said second fusion proteins are encoded by nucleotide sequences of a cDNA library. In still other embodiments step (a) comprises recombinantly expressing in a population of said cells a plurality of different said candidate molecules. In a further embodiment the method comprises isolating a nucleic acid encoding said first test protein from a cell in which said increase is detected in step (b).
A method of detecting one or more proteinxe2x80x94protein binding interactions comprising: (a) recombinantly expressing within a population of eukaryotic cells (i) first population of first fusion proteins comprising an eIF4G-like protein or a translationally active derivative thereof fused to a first test protein, wherein the first test protein varies among the population, (ii) a second population of second fusion proteins comprising an RNA-binding protein fused to a second test protein, wherein the second test protein varies among the population; wherein the cell comprises a DNA that is transcribed to produce a monocistronic or multicistronic RNA containing a heterologous protein-binding site in a region 5xe2x80x2 and adjacent to a reporter gene coding region, wherein said RNA-binding protein binds to said heterologous protein-binding site; and (b) detecting a cell that exhibits an increase in the amount of the protein encoded by said reporter gene coding region relative to said amount produced in the absence of one or both test proteins or relative to other cells in the population, wherein said increase indicates that the first and second test proteins in said cell bind to each other.
The invention provides a purified translationally active derivative of an eIF4G-like protein.
The invention provides populations of cells comprising a DNA molecule or a nucleic acid of described above.
The invention provides a pharmaceutical compositions comprising the DNA molecule or a nucleic acids described above in a pharmaceutically acceptable carrier.
The invention provides a method of treating a subject having a disease or disorder amenable to treatment by a protein comprising producing a therapeutically effective amount of said protein in said organism by a method comprising introducing into said subject: (a) a DNA molecule that is transcribed within the subject to produce a monocistronic or multicistronic RNA containing a heterologous protein-binding site in a region 5xe2x80x2 and adjacent to a coding region encoding said protein; and (b) a DNA molecule encoding a fusion protein such that the DNA molecule is expressed within the subject to produce said fusion protein, said fusion protein comprising an RNA-binding protein that binds to said heterologous protein-binding site, fused to an eIF4G-like protein or a translationally active derivative thereof. In one embodiment the fusion protein is expressed in the subject under control of an inducible promoter.
The invention provides a method of treating a subject having a disease or disorder amenable to treatment by a protein comprising (a) introducing into the subject: (i) a DNA molecule that is transcribed within the subject to produce a monocistronic or multicistronic RNA containing a heterologous protein-binding site in a region 5xe2x80x2 and adjacent to a coding region encoding said protein; and (ii) a DNA molecule encoding a fusion protein such that the DNA molecule is expressed within the subject to produce said fusion protein, said fusion protein comprising an RNA-binding protein that binds to said heterologous protein-binding site, fused to an eIF4G-like protein or a translationally active derivative thereof: and (b) administering the cell to the subject. In one embodiment the cell is a stem or progenitor cell.